Apparatus and methods for parallel processing of micro-volume liquid reactions

ABSTRACT

Disclosed herein are apparatuses and methods for conducting multiple simultaneous micro-volume chemical and biochemical reactions in an array format. In one embodiment, the format comprises an array of microholes in a substrate. Besides serving as an ordered array of sample chambers allowing the performance of multiple parallel reactions, the arrays can be used for reagent storage and transfer, library display, reagent synthesis, assembly of multiple identical reactions, dilution and desalting. Use of the arrays facilitates optical analysis of reactions, and allows optical analysis to be conducted in real time. Included within the invention are kits comprising a microhole apparatus and a reaction component of the method(s) to be carried out in the apparatus.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional ApplicationSer. No. 60/229,357, filed Feb. 18, 2000, the disclosure of which isincorporated herein by reference.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

[0002] Not applicable.

TECHNICAL FIELD

[0003] This invention is related to devices and processes for carryingout multiple simultaneous micro-reactions in liquid samples.

BACKGROUND

[0004] Reactions that are conducted in solution such as, for example,chemical, biological, biochemical and molecular biological reactions,are frequently carried out within a chamber or other container. Suchchambers, or reaction vessels, are commonly made of glass or plastic andinclude, for example, test tubes, microcentrifuge tubes, capillary tubesand microtiter plates. Reaction chambers currently in use are notamenable for use with volumes below one microliter, due to problems suchas large head volumes in the reaction chamber leading to evaporativelosses of the reaction solution, and difficulty in adding and removingreaction mixtures from the reaction chamber.

[0005] Many types of biochemical reactions, for example, nucleic acidamplification, require temperature cycling. Many reaction chambermaterials are poor thermal conductors, thus there are time lagsassociated with changing the temperature of the reaction vessel andequilibration of a temperature change throughout the sample volume. Suchlags in temperature change and temperature equilibration lead to longercycle times, non-uniform reaction conditions within a single reaction,and lack of reproducibility among multiple reactions, both simultaneousand sequential.

[0006] It is often necessary to carry out a series of experiments on aset of identical samples. Usually this set of samples must be seriallyduplicated, either manually or by means of robotic liquid deliverysystems. These processes can be slow, as they depend on the total numberof samples to be duplicated and, if applicable, the speed of the robot.

[0007] Efforts to address the aforementioned problems have included theuse of robotics and the use of capillary thermal cyclers, e.g., theLight Cycler® (Idaho Technologies). See Wittwer et al. (1997a)BioTechniques 22:130-138, and Wittwer et al. (1997b) BioTechniques22:176-181. However, such methods and apparatuses still require samplevolumes of several microliters, involve difficult liquid handlingprocedures such as loading and emptying capillaries, and can involvedetection problems associated with capillary geometry and spacing.

[0008] Microarrays comprising an ordered array of biological molecules(e.g., peptides, oligonucleotides) on a solid surface are known. See,for example, U.S. Pat. Nos. 5,445,934; 5,510,270; 5,605,662; 5,632,957;5,744,101; 5,807,522 5,929,208 and PCT publication WO 99/19510. Whilethese are useful for analyzing multiple molecules under identicalconditions (e.g., hybridizing a plurality of different oligonucleotidesequences with a single probe or probe mixture), such a “chip” cannot beused for analysis of multiple samples under multiple experimentalconditions. Furthermore, such arrays are limited to analysis ofmolecules which can either be synthesized on the array substrate orcovalently attached to the substrate in an ordered array. In addition,molecules tethered to an array react with slower kinetics than domolecules in solution, and are sterically hindered in their interactionsresulting in altered reaction kinetics. Additionally, for arrays ofproteins and peptides, surface interactions affect the naturalconformation of proteins under investigation (MacBeath and Schreiber,Science, vol 289, pp. 1760-1763).

[0009] WO 99/34920 discloses a system and method for analyzing aplurality of liquid samples, the system comprising a platen having twosubstantially parallel planar surfaces and a plurality of through-holesdimensioned so as to maintain a liquid sample in each through-hole bymeans of surface tension. WO 00/56456 discloses a method for holdingsamples for analysis and an apparatus thereof includes a testing platewith a pair of opposing surfaces and a plurality of holes. WO 99/47922discloses vascularized perfused microtissue/micro-organ arrays. U.S.Pat. No. 5,290,705 discloses a specimen support for optical observationor analysis, the support comprising a disc-like member composed of arigid material and having at least one unobstructed hole extendingtherethrough.

[0010] Polynucleotides may be sheared through transfer methods such aspipetting. One method for reducing polynucleotide shear is to use pipettips with the tip ends cut off for polynucleotide transfer. Othermethods for reducing polynucleotide shear have been described in U.S.Pat. Nos. 6,147,198; 4,861,448; 5,599,664; 5,888,723; and 5,840,862.

[0011] There is a continued need for apparatuses and methods suitablefor microvolume liquid reactions. There is also a need for improvedmethods of transferring polynucleotides.

[0012] All references cited herein are hereby incorporated by referencein their entirety.

DISCLOSURE OF THE INVENTION

[0013] Disclosed herein are apparatuses for containing multiplemicro-volume samples and conducting multiple simultaneous micro-volumechemical and biochemical reactions in an array format, methods utilizingthe apparatuses, and kits containing the apparatuses.

[0014] The embodiments of the invention include, but are not limited to,the following.

[0015] An apparatus for containing multiple micro-volume liquid samplescomprising a substrate, wherein the substrate defines a plurality ofsample chambers, wherein each sample chamber: (a) extends through thesubstrate, (b) comprises one or more walls and an opening at each end,and (c) holds a sample such that the sample is in the form of a thinfilm such that a liquid sample present in one sample chamber does notintermix with a liquid sample present in another sample chamber; andwherein the sample chamber has a height to width ratio of less than 1:1,wherein the height of the sample chamber is measured from one face ofthe substrate to the other. The apparatus may comprise at least onecomponent of a reaction to be carried out in the apparatus. In oneembodiment the component is a reagent used in a nucleotide sequencingreaction. In another embodiment the component is one used in ahybridization reaction. In another embodiment, the apparatus issubstantially free from contaminating amplifiable polynucleotides.

[0016] In another aspect of the invention, an apparatus for containingmultiple microvolume liquid samples comprising a substrate is provided,wherein the substrate defines a plurality of sample chambers, whereineach sample chamber: (a) extends through the substrate, (b) comprisesone or more walls and an opening at each end, and (c) holds a samplesuch that the sample is retained in the apparatus through surfacetension and such that a liquid sample present in one sample chamber doesnot intermix with a liquid sample present in another sample chamber;wherein the apparatus is substantially free of contaminating amplifiablepolynucleotides; and wherein the apparatus comprises at least onereagent used in a polynucleotide amplification reaction to be carriedout in the apparatus. In one embodiment, the apparatus comprises atleast two reagents used in a polynucleotide amplification reaction to becarried out in the apparatus. In a preferred embodiment, the samplechamber has a height to width ratio of about 1:1, wherein the height ofthe sample chamber is measured from one face of the substrate to theother. The polynucleotide amplification reaction may be, for example, apolymerase chain reaction, a ligase chain reaction, or a rolling circleamplification reaction.

[0017] For apparatuses disclosed herein, the reaction component(s) mayoptionally be affixed to the solid substrate. In some embodiments thereaction component is affixed to the solid substrate by drying.

[0018] In a preferred embodiment, the substrate comprises hydrophobicregions; the hydrophobic regions are located on the substrate such thata liquid sample present in one sample chamber does not intermix with aliquid sample present in another sample chamber. In an embodiment of theapparatus, the hydrophobic regions are located on the upper and lowerfaces of the substrate such that the openings of at least one samplechamber from at least one adjacent sample chamber by a hydrophobicregion. The hydrophobic regions may also be located on the walls of thesample chambers. In some embodiments the hydrophobic region forms anannular ring along the wall of the sample chamber. In some embodimentsthe apparatus comprises two or more hydrophobic regions, each forming anannular ring along the wall of the sample chamber, and the hydrophobicregions define one or more annular non-hydrophobic rings therebetween.

[0019] In another embodiment of the apparatus the substrate can comprisean upper face and a lower face. A further refinement of this embodimentis wherein the through axes of the sample chambers are perpendicular toboth faces of the substrate. The sample chamber may also have the shapeof, for example, a right circular cylinder or a right polygonal prism.

[0020] Other embodiments of the invention are methods that are carriedout in a microhole apparatus. These methods include the following:

[0021] A method for simultaneously conducting a plurality ofmicro-volume reactions, the method comprising: (a) introducing aplurality of liquid samples into the sample chambers of a microholeapparatus, wherein the samples contain necessary reaction components;and (b) placing the apparatus into an environment favorable to thereaction; wherein the microhole apparatus comprises a substrate, whereinthe substrate defines a plurality of sample chambers, wherein eachsample chamber: (i) extends through the substrate; (ii) comprises one ormore walls and an opening at each end; and holds a sample such that thesample is in the form of a thin film such that a liquid sample presentin one sample chamber does not intermix with a liquid sample present inanother sample chamber; and wherein the sample chamber has a height towidth ratio of less than 1:1, wherein the height of the sample chamberis measured from one face of the substrate to the other.

[0022] The environment can be one to prevent evaporation, such as ahydrophobic medium or a humidified chamber. In some embodiments theapparatus is substantially free of contaminating amplifiablepolynucleotides. In some embodiments the reactions can be ligationreactions, primer extension reactions, nucleotide sequencing reactions,restriction endonuclease digestions, oligonucleotide synthesisreactions, hybridization reactions, and biological interactions. Thebiological interactions can be avidin-biotin interactions,streptavidin-biotin interactions, antigen-antibody interactions,hapten-antibody interactions and ligand-receptor interactions. In someembodiments the reaction component is affixed to the substrate. In someembodiments the results of the reactions are monitored. Monitoring canbe by a number of methods, including optical monitoring, massspectrometry and electrophoresis. Monitoring can be of the progress ofthe reactions during the course of the reactions. In some of the methodsof the invention one or more of the reactions are supplemented with oneor more reagents during the course of the reaction.

[0023] In another method of the invention utilizing a microholeapparatus is one for adding a component to a microvolume reaction. Themethod comprises the steps of: A method for adding a component to amicro-volume reaction, wherein the method comprises the steps of: (a)providing a first apparatus comprising a first sample chamber containinga reaction mixture; (b) providing a second apparatus comprising a secondsample chamber containing the component; and (c) bringing theapparatuses into proximity such that liquid contact is establishedbetween the first sample chamber and the second sample chamber; whereineach apparatus comprises a substrate, wherein the substrate defines aplurality of sample chambers, wherein each sample chamber: (i) extendsthrough the substrate; (ii) comprises one or more walls and an openingat each end; and (iii) holds a sample such that the sample is in theform of a thin film such that a liquid sample present in one samplechamber does not intermix with a liquid sample present in another samplechamber; and wherein the sample chamber has a height to width ratio ofless than 1:1, wherein the height of the sample chamber is measured fromone face of the substrate to the other. In another embodiment of themethod multiple components are added to a reaction, by providingadditional apparatuses, wherein each of the components is present in asample chamber of an apparatus. Another embodiment comprisessimultaneously adding a component to a plurality of micro-volumereactions wherein, in the method described above, the apparatuses arebrought into proximity such that liquid contact is established betweencorresponding sample chambers of the apparatuses. In a preferredembodiment the component is a nucleic acid.

[0024] A method for adding a nucleic acid to a micro-volume reaction isprovided, wherein the method comprises the steps of: (a) providing afirst apparatus comprising a first sample chamber containing a reactionmixture; (b) providing a second apparatus comprising a second samplechamber containing the nucleic acid; and (c) bringing the apparatusesinto proximity such that liquid contact is established between the firstsample chamber and the second sample chamber; wherein each apparatuscomprises a substrate, wherein the substrate defines a plurality ofsample chambers, wherein each sample chamber: (i) extends through thesubstrate; (ii) comprises one or more walls and an opening at each end;and (iii) holds a sample such that the sample is retained in theapparatus through surface tension and such that a liquid sample presentin one sample chamber does not intermix with a liquid sample present inanother sample chamber.

[0025] A method for introducing a liquid sample into a sample chamber isprovided, wherein the method comprises the steps of: (a) contacting anapparatus with a liquid solution; and (b) removing the apparatus fromthe solution; wherein the apparatus comprises a substrate, wherein thesubstrate defines a plurality of sample chambers, wherein each samplechamber: (i) extends through the substrate; (ii) comprises one or morewalls and an opening at each end; and (iii) holds a sample such that thesample is in the form of a thin film such that a liquid sample presentin one sample chamber does not intermix with a liquid sample present inanother sample chamber; and wherein the sample chamber has a height towidth ratio of less than 1:1, wherein the height of the sample chamberis measured from one face of the substrate to the other.

[0026] In another embodiment of the invention, a method for introducinga liquid sample comprising a nucleic acid into a sample chamber isprovided, wherein the method comprises the steps of: (a) contacting anapparatus with a liquid solution comprising a nucleic acid; and (b)removing the apparatus from the solution; wherein the apparatuscomprises a substrate, wherein the substrate defines a plurality ofsample chambers, wherein each sample chamber: (i) extends through thesubstrate; (ii) comprises one or more walls and an opening at each end;and (iii) holds a sample such that the sample is retained in theapparatus through surface tension and such that a liquid sample presentin one sample chamber does not intermix with a liquid sample present inanother sample chamber.

[0027] In another embodiment of the invention, a method for diluting asolution is provided, wherein the method comprises the steps of: (a)providing a first apparatus comprising a first sample chamber containingthe solution (b) providing a second apparatus comprising a second samplechamber containing a diluent; and (c) bringing the apparatuses intoproximity such that liquid contact is established between the firstsample chamber and the second sample chamber; wherein each of theapparatuses comprises a substrate, wherein the substrate defines aplurality of sample chambers, wherein each sample chamber: (i) extendsthrough the substrate; (ii) comprises one or more walls and an openingat each end; and (iii) holds a sample such that the sample is in theform of a thin film such that a liquid sample present in one samplechamber does not intermix with a liquid sample present in another samplechamber; and wherein the sample chamber has a height to width ratio ofless than 1:1, wherein the height of the sample chamber is measured fromone face of the substrate to the other.

[0028] In another embodiment of the invention, a method for diluting asolution comprising a nucleic acid is provided, wherein the methodcomprises the steps of: (a) providing a first apparatus comprising afirst sample chamber containing the solution which comprises a nucleicacid; (b) providing a second apparatus comprising a second samplechamber containing a diluent; and (c) bringing the apparatuses intoproximity such that liquid contact is established between the firstsample chamber and the second sample chamber; wherein each of theapparatuses comprises a substrate, wherein the substrate defines aplurality of sample chambers, wherein each sample chamber: (i) extendsthrough the substrate; (ii) comprises one or more walls and an openingat each end; and (iii) holds a sample such that the sample is retainedin the apparatus through surface tension and such that a liquid samplepresent in one sample chamber does not intermix with a liquid samplepresent in another sample chamber.

[0029] In preferred embodiments for the methods described above aplurality of solutions may be simultaneously diluted wherein theapparatuses are brought into proximity such that liquid contact isestablished between corresponding sample chambers of the apparatuses. Inanother preferred embodiment, steps (b) through (c) are repeated one ormore times using a new second apparatus containing fresh solvent at eachrepetition of step (b).

[0030] In another embodiment of the invention, a method for selectiveretention of a molecule in a first sample chamber is provided, whereinthe method comprises the steps of: (a) providing a first apparatus,wherein the first sample chamber contains a solution comprising themolecule and one or more additional solute molecules of higherdiffusibility; (b) providing a second apparatus comprising a secondsample chamber containing a solvent; (c) bringing the apparatuses intoproximity such that liquid contact is established between the firstsample chamber and the second sample chamber; and (d) removing theapparatuses from proximity; wherein each of the apparatuses comprises asubstrate, wherein the substrate defines a plurality of sample chambers,wherein each sample chamber: (i) extends through the substrate; (ii)comprises one or more walls and an opening at each end; and (iii) holdsa sample such that the sample is in the form of a thin film such that aliquid sample present in one sample chamber does not intermix with aliquid sample present in another sample chamber; and wherein the samplechamber has a height to width ratio of less than 1:1, wherein the heightof the sample chamber is measured from one face of the substrate to theother.

[0031] A method for selective retention of a nucleic acid in a firstsample chamber is provided, wherein the method comprises the steps of:(a) providing a first apparatus, wherein the first sample chambercontains a solution comprising the nucleic acid and one or moreadditional solute molecules of higher diffusibility; (b) providing asecond apparatus comprising a second sample chamber containing asolvent; (c) bringing the apparatuses into proximity such that liquidcontact is established between the first sample chamber and the secondsample chamber; and (d) removing the apparatuses from proximity; whereineach of the apparatuses comprises a substrate, wherein the substratedefines a plurality of sample chambers, wherein each sample chamber: (i)extends through the substrate; (ii) comprises one or more walls and anopening at each end; and (iii) holds a sample such that the sample isretained in the apparatus through surface tension and such that a liquidsample present in one sample chamber does not intermix with a liquidsample present in another sample chamber.

[0032] In a preferred embodiment of these methods, the method is usedfor desalting a solution. In another preferred embodiment, steps (b)through (d) are repeated one or more times using a new second apparatuscontaining fresh solvent at each repetition of step (b). In anotherpreferred embodiment, a plurality of solutions may be simultaneouslydesalted, wherein the apparatuses are brought into proximity such thatliquid contact is established between corresponding sample chambers ofthe apparatuses.

[0033] A method for parallel electrophoretic analysis of a plurality ofmicro-volume reactions is provided, wherein the method comprises: (a)conducting the reactions in a microhole apparatus; (b) placing theapparatus in contact with an electrophoresis medium; and (c) conductingelectrophoresis; wherein the apparatus comprises a substrate, whereinthe substrate defines a plurality of sample chambers, wherein eachsample chamber: (i) extends through the substrate; (ii) comprises one ormore walls and an opening at each end; and (iii) holds a sample suchthat the sample is retained in the apparatus through surface tension andsuch that a liquid sample present in one sample chamber does notintermix with a liquid sample present in another sample chamber. Theapparatus can be placed with one face in contact with theelectrophoresis medium. In another embodiment the electrophoresis mediumis contained within the sample chambers of one or more additionalapparatuses. In other embodiments, in the additional apparatuses thecorresponding sample chambers are aligned.

[0034] Another method for use in the invention comprises a method forpreparing a plurality of samples for mass spectrometric analysis,wherein the samples are placed in an apparatus that comprises asubstrate, wherein the substrate defines a plurality of sample chambers,wherein each sample chamber: (a) extends through the substrate; (b)comprises one or more walls and an opening at each end; and (c) holds asample such that the sample is retained in the apparatus through surfacetension and such that a liquid sample present in one sample chamber doesnot intermix with a liquid sample present in another sample chamber. Theanalysis can be conducted by matrix assisted laser desorption ionizationtime-of-flight (MALDI-TOF) spectrometry. These methods can be used todetect a genetic polymorphism, for example, a single nucleotidepolymorphism (SNP). Detection may consist of, for example, massdifferences due to a single base primer extension.

[0035] A method for mixing a plurality of micro-volume samples isprovided, the method comprising: (a) providing a first microholeapparatus comprising a substrate, wherein the substrate defines aplurality of sample chambers, wherein each sample chamber: (i) extendsthrough the substrate;(ii) comprises one or more walls and an opening ateach end; and (iii) holds a sample such that the sample is retained inthe apparatus through surface tension and such that a liquid samplepresent in one sample chamber does not intermix with a liquid samplepresent in another sample chamber; (b) providing a second microholeapparatus comprising a substrate, wherein the substrate defines aplurality of sample chambers, wherein each sample chamber:(i) extendsthrough the substrate;(ii) comprises one or more walls and an opening ateach end; and (iii) holds a sample such that the sample is retained inthe apparatus through surface tension and such that a liquid samplepresent in one sample chamber does not intermix with a liquid samplepresent in another sample chamber; and (c) bringing the apparatuses intoproximity such that liquid contact is established between more than onesample chamber from the first apparatus and a sample chamber in thesecond apparatus. In one embodiment, the holes in the first apparatusmay be smaller than the holes in the second, allowing more than one holefrom the first apparatus to simultaneously contact a single hole in thesecond apparatus.

[0036] A method for simultaneously conducting a plurality ofmicro-volume polynucleotide amplification reactions is provided, themethod comprising: (a) introducing a plurality of liquid samples intothe sample chambers of a microhole apparatus, wherein the samplescontain necessary polynucleotide amplification reaction components; and(b) placing the apparatus into an environment favorable to thepolynucleotide amplification reaction; wherein the microhole apparatuscomprises a substrate, wherein the substrate defines a plurality ofsample chambers, wherein each sample chamber: (i) extends through thesubstrate; (ii) comprises one or more walls and an opening at each end;and (iii) holds a sample such that the sample is retained in theapparatus through surface tension and such that a liquid sample presentin one sample chamber does not intermix with a liquid sample present inanother sample chamber; and wherein the apparatus is substantially freeof contaminating amplifiable polynucleotides. In one embodiment, theenvironment is selected from the group consisting of a hydrophobicmedium and a humidified chamber. In another embodiment, thepolynucleotide amplification reaction is a polymerase chain reaction. Inother embodiments, the polynucleotide amplification reaction is a ligasechain reaction or a rolling circle amplification reaction. In someembodiments, the results of the reactions are monitored. The results maybe monitored by, for example, optical monitoring, mass spectrometry andelectrophoresis. In another embodiment, the progress of the reactionsare monitored during the course of the reactions. In yet anotherembodiment, one or more of the reactions are supplemented with one ormore reagents during the course of the reaction. In a preferredembodiment, a reagent is affixed to the substrate. In another preferredembodiment, the analysis is used to detect a genetic polymorphism. Inyet another preferred embodiment, the analysis is used to analyze geneexpression levels.

[0037] Still other embodiments of the invention are kits containing themicrohole apparatuses described Supra, for the methods of the invention.These include the following.

[0038] A kit comprising an apparatus for containing multiplemicro-volume liquid samples comprising a substrate, wherein thesubstrate defines a plurality of sample chambers, wherein each samplechamber: (a) extends through the substrate, (b) comprises one or morewalls and an opening at each end, and (c) holds a sample such that thesample is in the form of a thin film such that a liquid sample presentin one sample chamber does not intermix with a liquid sample present inanother sample chamber; and wherein the sample chamber has a height towidth ratio of less than 1:1, wherein the height of the sample chamberis measured from one face of the substrate to the other, and furthercomprising a reaction component packaged in a suitable container. Thereaction component may be a reagent for performing a reaction selectedfrom the group consisting of, for example, ligation reactions, primerextension reactions, nucleotide sequencing reactions, restrictionendonuclease digestions, oligonucleotide synthesis, hybridizationreactions and biological interactions.

[0039] A kit comprising an apparatus for containing multiplemicro-volume liquid samples comprising a substrate is provided, whereinthe substrate defines a plurality of sample chambers, wherein eachsample chamber: (a) extends through the substrate, (b) comprises one ormore walls and an opening at each end, and (c) holds a sample such thatthe sample is retained in the apparatus through surface tension and suchthat a liquid sample present in one sample chamber does not intermixwith a liquid sample present in another sample chamber; wherein theapparatus is substantially free of contaminating amplifiablepolynucleotides, and further comprising a polynucleotide amplificationreaction component packaged in a suitable container.

[0040] In one embodiment of the above described kits, the reactioncomponent may be affixed to the substrate. In another embodiment, thekit may further comprise a hydrophobic substance to be used with theapparatus. The hydrophobic substance can be, for example, a hydrophobicfluid packaged in a suitable container and/or a hydrophobic cover. Thekit may also further comprise a chamber for maintaining the appropriateenvironmental conditions for a reaction to be carried out in theapparatus. The kit may also further comprise an apparatus for loadingthe samples into the sample chambers.

[0041] As will be apparent to one of skill in the art, any method ortechnique which requires parallel processing, display and/or storage ofmultiple micro-volume samples will be facilitated by the use of theapparatuses disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0042]FIG. 1 is a perspective view of one embodiment of the claimedapparatus, in the form of a microhole array.

[0043]FIG. 2 is a side view (cutaway) through one row of an exemplaryapparatus.

[0044]FIGS. 3A and 3B show side (cutaway) views of a single exemplarysample chamber.

[0045]FIGS. 4A and 4B show side (cutaway) views of exemplary samplechambers containing alternating hydrophobic and hydrophilic regions.

[0046]FIGS. 5A, 5B and 5C shows simultaneous loading of multipleindividual samples into discrete sample chambers by contacting anexemplary apparatus with an arrangement of liquid samples on ahydrophobic surface.

[0047]FIG. 6A, 6B and 6C are top views of exemplary apparatuses that canbe used for gel loading.

[0048]FIG. 7 is a top view of one embodiment of the claimed apparatus,wherein the apparatus is taped with aluminum tape.

MODES FOR CARRYING OUT THE INVENTION

[0049] General Methods

[0050] The practice of the invention employs, unless otherwiseindicated, conventional techniques in photolithography, chemicaletching, general machining, microfluidics, organic chemistry,biochemistry, oligonucleotide synthesis and modification, nucleic acidhybridization, molecular biology, microbiology, genetic analysis,recombinant DNA, and related fields as are within the skill of the art.These techniques are described in the references cited herein and arefully explained in the literature. See, for example, Maniatis, Fritsch &Sambrook, MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring HarborLaboratory Press (1982); Sambrook, Fritsch & Maniatis, MOLECULARCLONING: A LABORATORY MANUAL, Second Edition, Cold Spring HarborLaboratory Press (1989); Ausubel, et al, CURRENT PROTOCOLS IN MOLECULARBIOLOGY, John Wiley & Sons (1987 and annual updates); Gait (ed.),OLIGONUCLEOTIDE SYNTHESIS: A PRACTICAL APPROACH, IRL Press (1984);Eckstein (ed.), OLIGONUCLEOTIDES AND ANALOGUES: A PRACTICAL APPROACH,IRL Press (1991); Birren et al. (eds.) GENOME ANALYSIS: A LABORATORYMANUAL, Cold Spring Harbor Laboratory Press, 1999.

[0051] The disclosures of all publications and patents cited herein arehereby incorporated by reference in their entirety.

[0052] Definitions

[0053] The terms “plate” and “substrate” denote the solid portion of anapparatus.

[0054] A characteristic of a “thin-film” sample, as disclosed herein, isthat a sample is contained in a sample hole and remains therein throughthe action of surface tension and/or adhesion to the inner wall of thehole. Preferably, a thin film is a liquid sample in which the diffusiontime is no more than about four-fold greater, more preferably no morethan about three-fold greater, more preferably no more than abouttwo-fold greater, more preferably no more than about one-fold greater inone dimension that in any other dimension. Preferably, the temperatureconductance characteristics of a thin film sample are no more than aboutfour-fold greater, more preferably no more than about three-foldgreater, more preferably no more than about two-fold greater, even morepreferably no more than about one-fold greater in one dimension that inany other dimension. Most preferably, a thin film will have hydrodynamicand thermal properties equivalent to a solution contained in a rightcircular cylinder having a depth:diameter ratio of about 4:1, or morepreferably about 3:1 or less, about 2:1 or less, more preferably about1:1, even more preferably less than 1:1. Methods for measuringhydrodynamic properties, diffusion time and thermal conductancecharacteristics are well-known to those of skill in the art.

[0055] Two or more holes in an apparatus are denoted “correspondingholes” if they occupy the same relative position on two or moredifferent apparatuses such that, if the apparatuses are alignedface-to-face, the holes communicate with one another.

[0056] “Polynucleotide”, “oligonucleotide”, and “nucleic acid”, are usedinterchangeably herein to refer to polymers of nucleotides of anylength, and includes natural, synthetic, and modified nucleic acids.

[0057] “Substantially free of contaminating amplifiablepolynucleotides”, as used herein, is meant to indicate an apparatuswhich is substantially free from contaminating polynucleotides, such asDNA or RNA, which may interfere with the analysis. Such an apparatus issuitable for use in performing assays such as, for example,amplification reactions, e.g. PCR reactions, in which contaminatingamplifiable polynucleotides may coamplify along with the desiredamplification product(s), thus interfering with the analysis.

[0058] Apparatuses

[0059] Disclosed herein are apparatuses and methods for simultaneousparallel processing, display and/or storage of a plurality ofmicro-volume liquid samples, wherein an apparatus comprises a substratecontaining a plurality of micro-sample chambers. In one embodiment, thesample chambers are micro-through-holes, such that the apparatuscomprises an array of micro-through-holes in a substrate. The apparatuscan have any shape consonant with the purposes for which it is used. Inone embodiment, the apparatus is rectangular; however, triangular,circular and ovoid shapes, among others, are also useful. An array ofmicroholes, as disclosed herein, can be used, for example, as amicro-volume sample holder and/or to conduct multiple parallelreactions.

[0060] Microholes can be placed in any arrangement within a substratethat is suitable for the experimental purpose of the apparatus. In oneembodiment, holes are arranged in rows and columns on a rectangularsubstrate. The size and/or shape of an apparatus can vary, and isdesigned with the particular experimental use of the apparatus in mind.For example, if the apparatus is to be used for gel loading or if theproducts of a reaction conducted in the apparatus are to be analyzed bygel electrophoresis (see infra), the size and shape of the apparatus canbe designed to match that of a gel electrophoresis apparatus or samplecomb.

[0061] An exemplary embodiment of the apparatus is described withreference to FIGS. 1 and 2, wherein the apparatus 1 comprises an arrayof micro-through-holes 5 contained in a substrate 6, such as a plate,wafer, film or slide, such substrate in one embodiment beingsubstantially thin and planar, and having an upper surface 7 and a lowersurface 8. In one embodiment, one or more of the surfaces of thesubstrate are rendered hydrophobic so that liquid reaction mixturescontained in the micro-through-holes will, by force of surface tensionand adhesion, remain fixed therein. In another embodiment, the substrateis flexible. In yet another embodiment, the substrate is a curved plane.An exemplary use of the latter embodiments is to bend the substrate intoa cylinder and place a rotating optical scanner inside the cylinder tomonitor the reactions in the microholes.

[0062] Any size and/or shape of the sample chamber, that is consistentwith the retention of liquid therein through surface tension and iscommensurate with the use of the apparatus, can be chosen. In onepreferred embodiment, the sample chambers are in the shape of a rightpolygonal prism, for example, a right rectangular prism. Although, in apreferred embodiment, the sample chambers are in the shape of rightcircular cylinders with parallel walls, it is clear that the walls of asample chamber could be convex (i.e., bowed inward) or concave (i.e.,bulged outward). Additional sample chamber configurations will beapparent to those of skill in the art and, indeed, any shape of samplechamber consistent with the retention of liquid therein through surfacetension is useful. In one embodiment, the height of the hole is greaterthan about four times the width. In other embodiments, the height of thehole is less than or equal to about four times the width, less than orequal to about three times the width, less than or equal to about 2.5times the width, less than or equal to about two times the width, equalto about one times the width, less than one times the width, less thanor equal to about 0.5 times the width. In another preferred embodiment,the height is equal to or less than the width. In a preferredembodiment, the height of the hole is about 1 times the width. In thisembodiment, the sample chamber is a microhole having an aspect ratiowith a width 11 roughly equal to depth 12. See FIG. 3A for the case of acylindrical sample chamber. In additional embodiments, the width 11 isgreater than the depth 12. See FIG. 3B, again directed to the exemplarycase in which the sample chamber is a cylinder. Thus, in a preferredembodiment, the diffusion time across the height is equal to or lessthan the diffusion time across the width. For a hole having the shape ofa right circular cylinder, the height:width ratio can also be expressedas the ratio of depth to diameter.

[0063] The size of the holes is commensurate with the reaction volumeand can be varied by varying the width (or diameter) of the hole and/orthe thickness of the substrate (which effectively varies the height ordepth of the hole). Thus, the volume of a reaction which can becontained in a hole is a function of the height of the hole and thewidth of the hole. However, a hole can be loaded such that the liquidextends beyond the physical boundaries of the hole; in some cases thiswill be facilitated if the surface of the substrate surrounding theopenings of the holes comprises a hydrophobic material; in other cases,it will be accomplished by surface tension. In this fashion, a volume ofliquid which is greater than the volume of the hole can be accommodatedby a sample chamber. Conversely, a hole can be loaded with a volume ofliquid that is less than the volume of the hole, such that the liquidsample forms a biconcave film. Thus the shape of the sample can rangefrom a biconvex disc through a flat disc to a biconcave disc.Accordingly, sample volumes of less than about 10000 nl, preferably lessthan about 1000 nl, preferably less than about 500 nl, preferably lessthan about 100 nl, more preferably less than about 250 nl, morepreferably less than about 100 nl, more preferably, less than about 50nl can be reliably achieved. In one embodiment, sample volumes as low as5 nl are used. Thus, sample volumes contemplated range from about 1 nlto about 10000 nl.

[0064] Each sample chamber can contain an individual sample, or a samplechamber can contain multiple samples separated by hydrophobic regionsalong the wall of the sample chamber. Thus, in one embodiment, theentire inner wall of a sample chamber is hydrophilic and the samplechamber contains a single sample. In another embodiment, the inner wallof a sample chamber is hydrophobic. In another embodiment, hydrophobicregions are located on the walls of the sample chambers. In a furtherembodiment, a hydrophobic region forms an annular ring along the wall ofthe sample chamber. Such a hydrophobic ring can be used, for example, todivide a sample chamber into two regions. Dual-region sample chamberscan be used, for example, to temporarily isolate different reactioncomponents prior to mixing by, for example, physical agitation,insertion and optionally movement of a probe and/or heating (e.g.,interior laser heating). Such a configuration is useful in the practiceof methods such as, for example, hot-start PCR. Additional applicationsof such a configuration will be apparent to those of skill in the art.

[0065] In accord with this embodiment, FIG. 4A shows a schematic diagramof an example of a microhole sample chamber containing annularhydrophobic region 21 along its wall, separating hydrophilic regions 22and 23. In additional embodiments, the wall of a sample chambercomprises two or more hydrophobic regions, each forming an annular ringalong the wall of the sample chamber, thereby defining a plurality ofannular non-hydrophobic rings. One example is diagrammed in FIG. 4B,which shows a schematic diagram of an exemplary microhole sample chambercontaining annular hydrophobic regions 25 and 26 along its wall,interspersed with annular hydrophilic regions 27, 28 and 29.

[0066] Hydrophobic and/or hydrophilic regions along the wall of a samplechamber need not form a continuous 360° ring, nor need they be in theshape of an annulus or portion thereof. Arcs or spots of hydrophilicand/or hydrophobic regions can be present, as required by the particularuse of the apparatus. For example, a hydrophobic annular ring,separating two aqueous samples, can be interrupted by a smallhydrophilic arc, the presence of which facilitates eventual mixing ofthe samples. In another example, a small hydrophilic region can bepresent on the wall of the sample chamber such that an aqueous samplecan be concentrated and optionally dehydrated onto the small hydrophilicregion. This can facilitate mass spectrometric analysis of a sample, byconcentrating the sample into a small target for the laser that is usedto launch the sample for mass spectrometry.

[0067] Apparatuses comprising holes with hydrophilic and hydrophobicregions located on the walls of the sample chamber may be constructed,for example, by laminating plates comprising different materialstogether with, for example, epoxy. For example, a titanium plate withchemically etched holes can be laminated on both sides to plastic platesalso etched with corresponding holes, yielding an apparatus withmicroholes having an annular hydrophilic ring surrounded by annularrings of hydrophobic regions.

[0068] In one embodiment, the apparatus is a plate with holes passingthrough in a direction perpendicular to at least one face of the plate.In another embodiment, the faces are parallel to each other, and theradial axis (i.e., the through axis) of each hole is parallel to thewalls of the chamber and perpendicular to the faces of the plate. In amore preferred embodiment, the holes have the shape of right circularcylinders. See, for example, FIG. 1. In another preferred embodiment,they have the shape of a right polygonal prism. The holes can bearranged in any configuration that is suitable to an experiment, e.g.,an array of one or more rows and one or more columns, the array being asquare array of holes, a triangular array of holes or anotherconfiguration of holes. The surface of the plate can be prepared ortreated so that it repels water or other aqueous solutions, thusensuring that small volumes of sample which are deposited in the holeswill remain in the holes without the possibility of leakage or crosscontact with samples in other holes.

[0069] The spacing between the holes can be varied to accommodate thedensity and pattern of holes on the substrate, so long as mixing betweenadjacent sample chambers does not occur. The substrate can have fromabout 1 to about 10,000 microholes per apparatus. In preferredembodiments there are at least about 600 microholes per apparatus, morepreferably at least about 800 microholes per apparatus, and even morepreferably at least about 1000 microholes per apparatus.

[0070] Evaporation may be minimized by providing an evaporation coveringsheet on the planar surfaces of the substrate which covers thethrough-holes and retains vapors within the reaction chamber. Such asheet may be comprised of any material which does not interfere with thereaction contained in the chamber. Such a sheet may be hydrophobic innature and may by flexible, such as silicone rubber, or may besubstantially rigid such as a polymeric or glass cover slide, and maycomprise an adhesive substance. The cover is preferably optically clear.

[0071] The top and bottom surfaces of the substrate may contain raisedfeatures which form closed curves circumscribing the openings to some orall of the sample chambers contained therein. Such features can be usedin conduction with an evaporation retention sheet to improve thereliability of said sheet to prevent loss of vapor. Preferably suchraised features are very narrow such that under a moderate force a veryhigh pressure is maintained at the interface of said feature(s) and theadjacent evaporation sheet. Additionally, a single raised feature maycircumscribe more that one reaction chamber and thus allow communicationbetween all reaction chambers contained therein.

[0072] In a preferred embodiment, the apparatus is substantially free ofamplifiable contaminating polynucleotides, particularly for reactions inwhich contaminating amplifiable polynucleotides may interfere with thereaction, e.g. PCR. Preferably, the apparatus has less than 1000amplifiable contaminating polynucleotides per reaction chamber, morepreferably less than 10 amplifiable contaminating polynucleotides perreaction chamber, even more preferably less than 1 amplifiablecontaminating polynucleotides per reaction chamber. Contaminatingamplifiable polynucleotides may be eliminated from the apparatus by, forexample, γ-irradiation. The presence of contaminating amplifiablepolynucleotides may be detected by, for example by performing a controlPCR reaction with no polynucleotide sample; a control reaction whichyields polynucleotides indicates the presence of contaminatingamplifiable polynucleotides.

[0073] Substrates

[0074] Numerous materials and methods are available for designing anapparatus for multiple micro-volume liquid samples. Materials that canbe used for the substrate include, but are not limited to, titaniumsheet preferably treated to render the surface hydrophobic, glass plateswith chemically etched holes and silanated surfaces, plastics, teflon,synthetics, metals and ceramics. Techniques of electrodepositionmanufacturing, and printed-circuit board manufacturing processes canalso be used in the fabrication of the apparatus. In one embodiment, thesubstrate has a hydrophobic surface and each sample chamber hashydrophilic interior walls. In another embodiment the hydrophilic regionof the interior of at least one of the sample chambers extends to thesurface of the substrate and extends beyond the orifice defined by thesample chamber such that the area occupied by the extended portion issubstantially contained on the substrate surface and such that thehydrophilic region of one sample chamber does not contact thehydrophilic region of any other sample chamber. Such a hydrophilicregion may aid in loading aqueous reactions into the reaction chamber.

[0075] Titanium is bio-inert, hydrophilic, and can be chemically etchedto provide a dense array of holes in any pattern desired. It is alsovery durable and hence reusable. Photo-etched titanium substrates arealso useful for fabrication and are available, for example, fromTech-Etch, Plymouth, Mass.

[0076] Glass plates rendered hydrophobic by a surface treatment, forexample, by silanation are also suitable; since glass is hydrophilic andsilanation renders its surface hydrophobic. Silicon microfabrication isa suitable method for fabrication of apparatuses comprising extremelywell defined, high density arrays of sample chambers.

[0077] Advances in the field of printed-circuit (PC) board manufacturingcan be applied to the fabrication of the apparatus. Current printedcircuit board technology provides both miniaturization and costefficiency.

[0078] An additional process which can be used in the fabrication of anapparatus as disclosed herein is photolithographic electrodeposition.This technique involves slowly depositing metal ions (electroplating)onto a substrate in a photolithographically defined pattern. Thistechnology reliably produces through-holes of 1 μm diameter, and canproduce over 3 million holes per square inch. In one embodiment, thistechnology is used for fabrication of an apparatus comprising very highdensity microhole arrays. Photolithographically fabricated substratesare available, for example, from Metrigraphics, Wilmington, Mass.

[0079] Other techniques for fabrication known in the art may be used forconstructing the apparatuses of the invention, for example, lasermicromachining and microfabrication techniques.

[0080] Sample Delivery and Recovery

[0081] Delivery of samples and reagents to a microhole can be achievedmanually, or through the use of commercially available pipetting robots,such as those available from the Hamilton Company, Reno, Nev. and thePackard Instrument Co., Meriden, Conn. Currently-available pipettingrobots can reliably deposit sample volumes as small as 50 nl, and arobotic positional repeatability of 50 μm is common. Higher accuracy andrepeatability can be achieved through special design, such as by thecoupling of piezoelectric and mechanical translation devices (e.g.,Physik Instrumente, Costa Mesa, Calif.). For dispensing reagent volumesin the sub-nanoliter range, piezo-electric pipettors and ink-jetpipettors, for example, can be used.

[0082] Thus, reactions can be prepared in the microholes of theapparatus by any means commonly used for dispensing small liquid volumesinto small vessels including, but not limited to: (1) dispensing verysmall volumes of reagent directly into each hole, either manually or bymeans of a robotically controlled syringe, (2) immersing the entireapparatus, or a predetermined fraction thereof, directly into a reactionsolution, thereby acquiring a volume of reaction solution in each holethat has been immersed, and (3) dispensing, pouring over or flowing overthe surface of the substrate a volume of the reaction mixture. Inanother embodiment, reaction components are affixed within a samplechamber, for example, by placing a solution within the sample chamberand drying it, such that a solute is affixed to the wall of a samplechamber. Reaction mixtures and/or additional reagents are then added tothe chamber, re-solubilizing the affixed reaction component. Inpreferred embodiments, more than one, more than two, more than threereagents may be affixed to the wall of a sample chamber. For PCRreactions, for example, primers, probes, and/or buffer components can bepreaffixed to the sample chambers, allowing for faster preparationtimes.

[0083] After initial addition of reagent, subsequent reagent additionscan be conducted. Means for adding additional reagent to a microholeinclude, but are not limited to, the previously-described methods, aswell as: (1) direct dispensing, either manually or by means of, forexample, a robotically controlled syringe, (2) direct dispensing intothe reaction solution by a non-contact means such as a piezo-electricdispensing apparatus, (3) deposition of a vaporized solution of reagent,and (4) contact between two or more of the apparatuses, such thatmaterial in a hole from one apparatus is transferred wholly or in partto a hole in another apparatus. In one embodiment, transfer occursbetween corresponding holes in two or more apparatuses.

[0084] An additional exemplary method for sample delivery to samplechambers in an apparatus is shown schematically in FIG. 5. In thisembodiment, samples 31 are arranged on a hydrophobic surface 32 in apattern that matches the pattern of holes 33 in an apparatus 34. FIG.5A. The apparatus 34 is then brought into proximity with the hydrophobicsurface 32, such that the samples 31 contact the holes 33 in theapparatus 34. Figure SB. The apparatus is then withdrawn from proximitywith the surface, the holes 33 now containing samples 31 (Figure SC).

[0085] The process of liquid transfer from one apparatus to another canalso be used to make several copies of a single setup platesimultaneously. For example, if a group of microhole array plates arestacked, one on top of another, and liquid samples are introduced intothe top or bottom plate, the samples will wick through the entire stack,thereby generating a series of plates having identical sampleconfigurations.

[0086] Furthermore, contents of a sample chamber, or of an entireapparatus, can undergo dilution, particle size selection, selectiveretention of a molecule in a sample chamber, desalting, reagentaddition, or another chemical modification by bringing the apparatus, ora portion thereof, in liquid contact with one or more additionalapparatuses, the additional apparatus(es) prepared in such a way thatcontact between apparatuses will, by chemical diffusion from one samplechamber on one apparatus to the another sample chamber on an adjacentapparatus, achieve the required sample modification. See infra.

[0087] After reaction assembly in an apparatus is complete, theapparatus can subsequently undergo chemical processing and/or incubationin a thermally-controlled environment. A concern when dealing withminute aqueous samples, such as are present in the sample chambers ofthe apparatus, is evaporation. One way in which this problem can bemitigated is by conducting incubation in a high-humidity environment.For example, various types of water vaporizers are readily available andcan be easily integrated into a laboratory device to provide ahumidified chamber. Other methods of reducing evaporation include, butare not limited to, placing the apparatus in a humidified chamber,maintaining the atmosphere of an open apparatus at saturated vaporpressure, and periodic addition of water to the reactions by means of apiezo-electric or other dispenser in a manner which counteracts theevaporation rate.

[0088] Alternatively, the apparatus can be immersed in a hydrophobicmedium such as, for example, a bath of an inert liquid that isessentially non-miscible with water and which essentially does not reactwith the substrate nor essentially perturb the reaction(s) contained inthe sample chamber(s). In many applications an example of a suitablehydrophobic medium is oil, for example, mineral oil or silicone oil.

[0089] Reactions contained in the sample chambers of an apparatus can bethermally cycled in such a way as to carry out, for example,amplification reactions such as a polymerase chain reaction (PCR) or DNAsequencing reactions such as, for example, chain-termination sequencingand cycle sequencing. In such thermal cycling reactions, evaporation ofthe sample can be minimized by submerging the sample in a hydrophobicmedium such as, for example, a bath of hydrophobic medium or othersuitable fluid which is maintained at a temperature appropriate for thechemical reaction, by coating the apparatus with a layer of hydrophobicfluid, or by overlaying the samples with a hydrophobic fluid. Forexample, addition of samples to sample chambers can be followed bydirect addition of a film of oil or other hydrophobic fluid to eachsample using a pair of robotic pipets; one filled with a sample and theother filled with the hydrophobic fluid.

[0090] If, for example, an oil bath is used for temperature control,temperature cycling can be achieved by thermally cycling the bath, or byrobotically moving an apparatus from one bath to another bath held at adifferent temperature. Alternatively, thermal cycling of reactionscontained in an array of microholes can be carried out in a humidifiedenvironment, maintained by sealing the array between one or twoevaporation barriers (e.g., silicone sheets or Parafilm®), and placingthis sandwiched array on a thermoelectric heating block, or between twothermoelectric heating blocks. In using an evaporation barrier such as asilicone sheet, the presence of narrow raised features circumscribingeach sample chamber or a set of sample chambers to aid in preventing theloss of vapor by providing a very tight seal around each hole.

[0091] Means for dispensing water to counter evaporation can also beused to dispense other reagents or chemicals of interest, mixed in asolution such that evaporation is countered. In this way, chemicalassays can be carried out in real time and the evolution of the assaycan be directed via feedback from the assay in progress. To provide butone example, optical data obtained by placing the apparatus on anoptical detector, such as a CCD or fiber optics cable, can be input to acomputer which automatically adjusts reagent dispensers and guides themto dispense a precise amount of reagent into each sample chamber.

[0092] Advantages

[0093] The apparatuses disclosed herein, when used, for example, in abiochemical reaction format, provide the advantages of: (1) highdensity, (2) high throughput, (3) ease of handling, (4) performance ofvery low-volume reactions (5) rapid thermal cycling, and (6)advantageous optical access of the samples. In embodiments in which theapparatus is moved from one thermal environment to another, thethin-film nature of the samples ensures very rapid thermal equilibrationtime. In an alternative embodiment in which an apparatus is heldstationary in a thermoelectric (Peltier) device and the temperature ofthe device is changed, apparatuses having a planar symmetry allow twothermoelectric devices to be used, one on each side of the apparatus.This provides a sample ramp speed at least twice that obtained when asingle Peltier device is used, as well as finer control of thetemperature profile within the sample.

[0094] Further benefits of the microhole array format of the apparatusinclude the ability to transfer liquids from one sample chamber in afirst apparatus to another sample chamber in a second apparatus simplyby bringing two apparatuses in close enough proximity to allow thecontents of two sample chambers to touch. Upon physical contact the twosamples will diffuse together. This technique allows sample mixing,sample dilution and diffusion-based molecular separations e.g.,de-salting. The contents of a sample chamber in a first apparatus can betransferred, in whole or in part, to a sample chamber in a secondapparatus.

[0095] For sample mixing, two apparatuses are brought into contact, asabove, such that the liquid contents of one or more pairs of samplechambers come into liquid contact, wherein one member of each pair ofsample chambers is present in a first apparatus and the other member ispresent in a second apparatus. Mixing of three or more samples, usingthree, four, etc. apparatuses is also possible, as will be evident toone of skill in the art. This is particularly advantageous when beingused to transfer nucleic acids, such as DNA and RNA, from one apparatusto another. Nucleic acids are easily sheared by methods such aspipeting, and this method allows for the transfer of nucleic acidswithout the need for pipetting.

[0096] For sample dilution, a first apparatus containing one or moresamples is contacted with a second apparatus containing diluent, suchthat liquid contact is achieved between one or more sample chambers inthe first apparatus and one or more sample chambers in the secondapparatus, and the apparatuses are allowed to remain in contact for aspecified time. In this case, all components in the sample are diluted,with the degree of dilution depending on the time of contact between thefirst and second apparatuses. If all samples in a first apparatus are tobe diluted, the first apparatus need not be contacted with a secondapparatus, but can simply be contacted with a pool of diluent.

[0097] Selective retention of a molecule in a sample chamber, dependenton diffusion-based separation of low molecular weight molecules frommolecules of high molecular weight, is possible using the apparatuses asdisclosed herein. For example, a sample contained in a microhole isdesalted by repeatedly touching the sample, for a short time, to a bath(or to a microhole of another apparatus) containing a solution of verylow salt concentration. Since very small molecules (such as salts)diffuse very rapidly (approximately 60 μm per second), while largermolecules take much longer to diffuse (e.g., a 2 kilobase nucleic acidhas a diffusion rate of approximately 6 μm per second, see, for example,Smith et al. (1996) Macromolecules 29:1372-1373), an overall reductionin salt concentration of the sample is achieved.

[0098] In one embodiment, two apparatuses having identical patterns ofsample chambers are contacted so as to bring corresponding samplechambers into contact. Two (or more) sample chambers are “corresponding”if they are located in the same position on different apparatuses (i.e.,if each apparatus comprises an array of microholes, correspondingmicroholes occupy the same location in the array). In additionalembodiments for selective, diffusion-based molecular retention,desalting, dilution and/or reagent addition, contact betweencorresponding sample chambers is not required. For example, a singlesample chamber in a first apparatus can be contacted with multiple,different sample chambers of a second apparatus. In a separateembodiment, subsets of chambers in a first apparatus are contacted withsets of chambers in a plurality of additional apparatuses.

[0099] Another advantage of the apparatus format of the invention is theability to minimize shear when loading a microhole array with a nucleicacid. Nucleic acids, such as DNA and RNA, are easily sheared by transfermethods such as pipetting. The apparatus of the invention may be loadedwith a solution comprising a nucleic acid simply by contacting theapparatus with a liquid solution, for example, contacting the apparatuswith a tray containing the solution of interest (e.g., “dip loading”).

[0100] In some embodiments, formation of a thin film by a sample, whenit is contained in a sample chamber of the apparatus, results in a ratioof surface area to volume that facilitates optical analysis of thesample, either continuously during the reaction period or at one orseveral predetermined time points. Because photons pass through aminimum fluid volume in the thin film, more efficient detection of lightabsorption and emission (e.g., fluorescent, chemiluminescent) by asample is possible. Furthermore, because of these favorable opticalproperties, progress of multiple reactions can be monitored in real time(i.e., during the course of the reaction). For example, in reactionsthat generate an optical signal, e.g. a colored, fluorescent, orluminescent product, reaction progress can be monitored in real time forinstance using multiple optical detectors aligned with the samplechambers, fiber optics, a detector that scans across the apparatus, or awhole-apparatus imager. Compared to analysis of micro-volume reactionsin capillaries, the apparatuses disclosed herein allow improved opticalanalysis that is not prone to either refractive effects from capillarywalls or optical cross-talk between neighboring capillaries.

[0101] Applications

[0102] Apparatuses as disclosed herein can be used for holding andarraying any type of liquid micro-volume sample. They can also be usedfor performing any type of biochemical or molecular biological reactionknown to one of skill in the art including, but not limited to,nucleotide sequencing (e.g., chain-termination sequencing, cyclesequencing), amplification reactions (e.g., polymerase chain reactions),transcription, reverse transcription, restriction enzyme digestion,ligation, primer extension, other enzymatic reactions and biologicalinteractions (such as, for example, avidin-biotin, streptavidin-biotin,antibody-antigen and ligand-receptor interactions). In general, any typeof enzyme-mediated reaction can be performed in the apparatus. Inaddition, multiple micro-volume hybridization reactions can be conductedin the apparatus. In one embodiment, an apparatus is used for very highthroughput analysis of chemical samples; for example, in combinatorialchemistry. Several examplary applications are disclosed below, includingthose in the Examples, and additional applications are known to those ofskill in the art. Use of the apparatuses disclosed herein will beespecially useful in the field of genetic analysis, for techniques suchas polymorphism detection (see infra).

[0103] Amplification Reactions

[0104] Very high throughput of small volume amplification reactions,such as polymerase chain reactions, ligase chain reactions, rollingcircle amplification, and “Taqman®” hydrolyzable probe assays isobtained using, for example, an apparatus containing an array ofmicroholes. The ability to perform a large number of individualreactions, each in a very small volume, obviates the need for multiplexPCR (in which several different genomic loci are amplified in a singlereaction) and avoids the technical difficulties inherent in thatstrategy. Alternatively, low-number multiplex reactions can be carriedout in a microhole format, compounding the benefits of this technology.

[0105] In many applications of PCR, recovery of an amplification productis desirable. Problems with recovery of amplification products usingmethods of the prior art are related to the elevated temperatures usedfor most amplification reactions, necessitating the use of an oiloverlay to prevent evaporation of the reaction mixture. In these cases,the presence of oil can interfere with recovery of the amplificationproduct(s), for example, making it difficult to aspirate a microvolumesample. Methods which do not require the use of oil (e.g., conductingamplification reactions in capillaries) still present problems withfluid manipulation.

[0106] This problem can be addressed by use of apparatuses as disclosedherein. For example, a porous hydrophobic membrane that is preferablyessentially non-reactive (such as a teflon membrane filter) can be usedin conjunction with an apparatus. In this embodiment, an apparatuscontaining a plurality of reactions is immersed in an oil bath forconducting a high-temperature reaction, removed from the bath, andtouched to the porous hydrophobic membrane. The hydrophobic medium willreadily wet the hydrophobic membrane and flow into it, whereas anaqueous reaction solution will be repelled. Subsequent removal of theapparatus from the hydrophobic membrane leads to the formation ofdiscrete drops of aqueous solution resting thereon. These aqueous dropscan be readily accessed. Alternatively, direct pipetting of an aqueousreaction solution from beneath an hydrophobic substance layer onhydrophobic surface is possible with accurate and reliable robotics.

[0107] Moreover, for certain applications, the presence of a hydrophobicsubstance is not a hindrance. For example, when an apparatus is used forloading reaction products into an acrylamide gel (see infra), theapparatus (optionally having been used as a reaction sheet) can beplaced onto the top of a gel and overlayed with upper reservoir buffer.After addition of buffer, the hydrophobic substance separates from thebuffer layer, thereby separating from the gel samples.

[0108] Very High Throughput PCR

[0109] High throughput PCR is readily achieved using, for example,microhole arrays by dispensing template, reagent and primer pairs toeach microhole. Typically, two of these steps are combined: for instancethe template and reagent (enzyme, buffer, etc.) are combined in a mastermix and the master mix is loaded simultaneously into all microholes bydipping an apparatus into a solution of master mix or by spraying asolution of master mix over an apparatus such that the solution entersthe sample chambers. A significant increase in throughput is achieved bypre-affixing oligonucleotide primers and probes to each chamber, or bypre-synthesis of all primer pairs in a microhole array. In this case,reactions can then be assembled simply by immersing a pre-synthesizedmicrohole array plate into a bath of master mix.

[0110] Technologies for pre-synthesis of primers on the apparatusinclude: standard phosphoramidite and photo-phosphoramidite chemistries.Standard phosphoramidite chemistry is used for most oligonucleotidesynthesis operations when it is necessary to recover the oligonucleotidein solution for later use. See, for example, U.S. Pat. Nos. 4,415,732;4,458,066; 4,500,707; 4,973,679; and 5,153,319. Photo-phosphoramiditechemistry, for synthesizing oligonucleotides on a solid substrate forlater use on that substrate, has been disclosed, for example, in U.S.Pat. Nos. 5,445,934; 5,510,270; and 5,744,101; and PCT publication WO99/19510.

[0111] Using photo-phosphoramidite chemistry, substrates containing upto 10,000 discrete oligonucleotides can be obtained. In one embodiment,an output array is controlled by an array of micro-mirrors and opticalelements and is highly flexible. Singh-Gasson et al. (1999) NatureBiotechnology 17:974-978. Thus, this technique is suitable for formingarrays for use in highly parallel processing and can be adapted forsynthesizing oligonucleotides in, for example, an array of microholes.

[0112] Cycle Sequencing

[0113] Small volume cycle sequencing reactions can be performed in ahigh throughput setting, in a fashion similar to the PCR application,supra. An additional benefit of the technology disclosed herein is theability to perform sequencing reactions directly on the comb or platethat will be used for loading the reaction product onto the sequencingapparatus. For instance, for using standard slab gel electrophoresissequencing apparatus, reactions are performed in a 1-dimensional arrayof holes which has been pre-formed on a gel loading comb. See, forexample, Erfle et al. (1997) Nucleic Acids Res. 25(11):2229-2230.Sequencing reactions are assembled in the array of holes on the comband, after completion of thermal cycling, the reaction product isdirectly loaded onto a gel for electrophoresis. FIG. 6 depicts severalembodiments of this type of array. FIG. 6A shows an apparatus 40 inwhich the sample chambers 41 are located close to one edge 42 of theapparatus. FIG. 6B shows a portion of an apparatus 50 in which thesample chambers 51 communicate with the exterior of the apparatus. FIG.6C shows a portion of an apparatus 60 in which the sample chambers 61communicate with the exterior of the apparatus via channels 62.Additional configurations of the apparatus suitable for gel loading willbe apparent to those of skill in the art.

[0114] Endonuclease Reactions

[0115] Standard room temperature or elevated temperature restrictionendonuclease digestions can be performed using the disclosedapparatuses. An endonuclease reagent master-mix is loaded into eachsample chamber, either by automated pipetting means or by immersing, forexample, a microhole array into a bath of reagent. Subsequently,individual samples of, for example, nucleic acid and/or restrictionendonuclease, can be loaded into each well using an automated pipettoror other means. After the reactions are assembled the apparatus isincubated at a temperature appropriate for the assay. If the durationand temperature of the incubation is such that evaporation of thesamples may be a problem, the incubation can take place in a humidifiedchamber or under a hydrophobic substance to counter or mitigate theeffects of evaporation. See supra.

[0116] Biological Interactions

[0117] The strong biochemical interaction between biotin andstreptavidin (or avidin) has made these molecules useful for bindingassays. For example, incorporation of a biotin-labeled nucleotide into apolynucleotide, and subsequent capture of the polynucleotide withstreptavidin, is a common method for isolating a specific polynucleotidesequence. A biotin-streptavidin capture is easily performed using thedisclosed apparatus by simply touching an apparatus, optionallycontaining biotin-labeled samples, to a plate or substrate which hasstreptavidin bound to its surface. The plate can be a membrane,microscope slide, cover slip, or another microhole array withstreptavidin bound to the inner wall of the sample chambers.

[0118] Other pairs of interacting molecules can also be used in asimilar fashion. Examples include, but are not limited to,antigen-antibody, hapten-antibody, sugar-lectin, and ligand-receptor.

[0119] Oligonucleotide Synthesis

[0120] In another embodiment for performing chemical reactions, anapparatus such as a microhole array is used as a miniatureoligonucleotide synthesizer, utilizing standard phosphoramiditechemistry and electrical addressing. It is a straightforward extensionof microfabrication technologies as used in integrated circuitproduction to design a microhole array in which each hole can beindividually charged or uncharged. See, for example, U.S. Pat. Nos.5,605,662; 5,632,957; and 5,929,208 for related techniques used in theconstruction of a microarray. Using such techniques, a differentoligonucleotide can be synthesized at each hole in a multi-step process.At each step, the array is exposed to a nucleotide monomer, and thesites on the array containing oligomers to which that monomer is to beadded are electrically addressed so as to direct the monomer to thosesites.

[0121] Such a device would have the multiple advantages of producingoligonucleotides in a predetermined location as well as producingsmaller amounts of oligonucleotide required for a particular reaction,leading to increased economy and efficiency.

[0122] Genetic Analysis

[0123] An important result of the efforts to determine human (and other)genome sequences is the availability of a vast pool of genetic data (inthe form of DNA sequence) which can be subjected to a multitude ofgenetic analyses. The results of the various genetic analyses can beapplied to diagnostic, pharmacogenomic and therapeutic applications, toname but a few. One particularly valuable form of genetic informationthat is available through the analysis of DNA sequence is geneticpolymorphism. Polymorphism can be due to insertion, deletion,translocation, transposition and/or tandem repetition of particularportions of a sequence, or to single- or multiple-nucleotide changes atparticular positions within a sequence.

[0124] Many methods known in the art can be used to determine thepresence of a genetic polymorphism. For example, insertions anddeletions, as well as some types of transposition and translocation, canbe detected by restriction fragment length polymorphisms (RFLPs).

[0125] Another method for determining the presence of a polymorphism isby analysis of tandem repeat lengths in minisatellite DNA. Thistechnique involves restriction enzyme digestion and blot hybridization,and/or STRP analysis, which involves PCR, gel electrophoresis or primerextension and mass spectrometry. See, for example, Birren et al. (eds.)“Genome Analysis: A Laboratory Manual” Cold Spring Harbor LaboratoryPress, 1999, esp. Volume 4.

[0126] Polymorphisms resulting from a single nucleotide change (“SNP”)may or may not result in a change in the size of a restriction fragment.A multitude of additional techniques, known to those of skill in theart, are available for the detection of SNPs. These include, but are notlimited to, denaturing gradient gel electrophoresis, single-strandconformation polymorphism analysis, heteroduplex analysis, temperaturegradient gel electrophoresis, cleavase-fragment length polymorphism,denaturing HPLC, chemical cleavage of mismatch, carbodiimidemodification, enzymatic cleavage of mismatch,uracil-N-glycosylase-mediated T scan, direct nucleotide sequencing, DNAchip resequencing, allele-specific primer extension, oligonucleotideligation assay, randomly amplified polymorphic DNA analysis(“RAPD”),fluorescence energy transfer dye terminator incorporation assay (“FRETTDI”), dye-labeled oligonucleotide ligation assay (“DOL”), Taqman® withallele-specific oligonucleotides, randomly amplified polymorphic DNAs,and analysis by the Invader® (Third Wave Technologies) technique.Additional methods of polymorphism analysis are known to those of skillin the art. See, for example, Birren et al., supra.

[0127] In an embodiment of the invention, genetic polymorphism analysiscan be carried out as described supra, in the apparatuses disclosedherein, which will thus be useful in these types of genetic analysis.

[0128] A method for SNP determination is by single base primerextension. In this technique, a primer is annealed to a polynucleotidethat is to be tested for the presence of a SNP. The sequence of theprimer is chosen such that the 3′-terminal nucleotide of the primer isadjacent to the site that is to be tested for the presence of the SNP.This template-primer complex is used for the preparation of fourseparate primer extension reactions, each containing only a singlenucleotide. The reaction(s) in which extension occurs provides thesequence of the site being tested for the presence of the SNP. See, forexample, U.S. Pat. No. 6,004,744.

[0129] Although extension can be assayed by DNA sequencing techniques,alternative assays are known in the art. One alternative assay forextension measures increases in molecular weight by mass spectroscopy,for example, matrix assisted laser desorption ionization time-of-flight(MALDI-TOF) spectrometry. Primers that have been extended by a singlenucleotide, having a higher molecular weight, will be distinguished fromunextended primers when analyzed by MALDI-TOF or other forms of massspectrometry.

[0130] The apparatuses disclosed herein are useful in MALDI-TOF, andother types of mass spectrometric analyses, because transfer ofextension products to a mass spectrometry preparation platform can beachieved by touching rather than pipetting. Consequently, multiplesamples can be transferred simultaneously. Rapid sample preparation formultiple mass spectrophotometric analyses is accomplished by dehydrationconcentration of a sample on a hydrophilic region of an apparatus. Thehydrophilic region can be located, for example, on the wall of a samplechamber.

[0131] The discovery of SNPs is efficiently accomplished by directnucleotide sequence determination. The apparatuses disclosed hereinprovide an ideal route to SNP discovery by facilitating high-throughputnucleotide sequence determination.

[0132] Sandwiched Reactions

[0133] Chemical and biochemical reactions can be assembled by placingapparatuses containing individually pre-filled sample chambers adjacentto each other in such a way that corresponding samples in differentapparatuses attain physical contact and the contents of thecorresponding samples mix spontaneously. In one embodiment, the samplechambers in an apparatus are pre-loaded and allowed to dry so that thecontents of the chambers (e.g., oligonucleotide primers) are in a stateof dehydration. When such a dehydrated apparatus is brought into contactwith an apparatus whose sample chambers are loaded with an aqueousreaction component, the dehydrated component(s) will be re-hydrated.This method can be practiced with several apparatuses at a time,allowing complex reactions to be assembled. A further advantage is thatthis method minimizes effects of evaporation that occur during reactionassembly, because all sample chambers are re-hydrated simultaneouslywhen a stack of, for example, microhole arrays is assembled. Reactionscan be conducted in a plurality of apparatuses arranged in a stacked orsandwiched configuration.

[0134] Addition of Reagents During Chemical Processing

[0135] Reagents can be added to reactions in progress in an apparatus,during incubation, by means of a piezo-electric dispenser or othercommon apparatus. In this way, the effects of evaporation can becountered by the addition of water; alternatively, chemicals and/orenzymes can be added to a reaction in progress. Using this method,reactions can be optimized as they proceed (i.e., in real-time).

[0136] Library Display and Assay

[0137] The apparatus of the invention can be used for creating anddisplaying libraries, for example, libraries of cells. For example, thesample chambers can comprise an adherent surface, suitable for cellgrowth, such as plastic, polystyrene and optionally polylysine. Adifferent cell, cell strain or cell type can be applied to each samplechamber and the substrate placed under conditions suitable for cellgrowth; for example, the substrate is immersed in culture medium in aCO₂ incubator at 37° C. After a period of cell growth, the substrate isremoved from the growth conditions and subjected to conditions thatresult in cell lysis and fixation of cellular macromolecules within oradjacent to the sample chambers. The substrate can then be subjected to,for example, restriction enzyme digestion, hybridization and/oramplification analysis to determine the presence of a particular targetmacromolecule in a particular cell, for example.

[0138] Real-Time Analysis of Reactions

[0139] Optical monitoring of the reactions contained in the samplechambers of the apparatus can be achieved in several ways. For example,an array of microholes containing a plurality of completed reactions canbe placed in direct contact with a CCD array or fiber optics bundle, orcan be loaded into an optical reading apparatus. For light-emittingreadouts, such as fluorescence or chemiluminescence, a benefit of themicrohole array is that there may be no plastic or other material toobscure or diffuse the emitted light and potentially generateautofluorescence. For thermal cycling reactions contained in ahydrophobic medium such as a bath of a hydrophobic substance, it ispossible to monitor the optical activity of the reaction continuously.The hydrophobic substance itself can optically couple the reaction to afiber optic bundle immersed in the hydrophobic substance which directsemitted light to a CCD array for quantitative detection. Such a deviceallows very high parallel processing of real-time assays such as PCR andTaqman®. Using fiber optic bundles capable of carrying hundreds ofthousands to millions of individual fibers, it is possible to monitormillions of amplification reactions simultaneously and in real-time on asingle apparatus such as a microhole plate.

[0140] High Throughput Sequencing

[0141] In addition to providing significant advantages in cyclesequencing, as described above, the use of the disclosed apparatuses,such as microhole arrays, for chemical reactions provides advantages instandard nucleotide sequencing operations as well. For example, it ispossible to design a microhole sheet that is thin enough to fit betweenthe glass plates of a standard polyacrylamide slab gel. The sheet hasmicroholes arranged in a straight line array with each microholecontaining a sub-microliter volume of a chain termination sequencingreaction. See, for example, FIG. 6A. The sheet is placed into a constanttemperature or thermal cycling apparatus for processing of thesequencing reaction, and is then transferred to the top of apolyacrylamide gel or other electrophoresis medium, with one edge of thesheet contacting the electrophoresis medium. In one embodiment, thesheet contains partial holes along its perimeter into which reactionmixtures are dispensed and reactions are conducted, and from whichreaction mixtures are applied directly to a gel by the method describedabove. A partial hole is one which is not completely enclosed by thesubstrate, such that up to about 180° of the diameter of the hole is notenclosed by the substrate, i.e., up to 180° of the hole diameter is opento the exterior of the substrate. See, for example, FIG. 6B.Alternatively, a hole can communicate with the exterior of the substratethrough a thin channel in the substrate. See, for example, FIG. 6C.

[0142] For sequencing techniques in which the termination productscorresponding to a particular one of the four nucleotides are labeledwith a chromophore or fluorophore specific to that nucleotide, it ispossible to combine the four base-specific reactions for analysis on asingle gel lane. In this case, four arrays, each containing a differentone of the four sequencing reactions in a corresponding hole, arestacked, and the stack is placed in contact with a sequencing gel suchthat the samples enter the gel upon provision of an electric current.Alternatively, the four plates are stacked so that the four sequencingreactions mix and the mixture equilibrates throughout the stack. Thenone of the plates is removed from the stack and placed in contact withthe gel.

[0143] Use of an apparatus such as a microhole sheet for gel loading hasmany advantages. The very small reaction sizes available with the sheetsresults in reduced reagent usage and consequent cost savings.Additionally, a sheet can be pre-loaded with hundreds of sequencingreactions, rather than the current limit of 96 samples per gel, therebysignificantly expanding the capacity and throughput of current gel-basedsequencing techniques and exceeding those of capillary sequencinginstruments.

[0144] Additional Electrophoretic Applications

[0145] In another embodiment of the use of the disclosed apparatuses forelectrophoresis, a microhole array containing a plurality of samples isplaced such that one face of the array is in contact with anelectrophoresis medium. In this way, several rows of samples can besimultaneously transferred to the electrophoretic medium to provide athree-dimensional electrophoretic analysis. Detection of samples duringand/or after electrophoresis is accomplished, for example, byfluorescence. In one embodiment, a molecule which becomes fluorescentupon DNA binding (such as ethidium bromide, acridine orange or SYBRgreen, for example) is present in the electrophoretic medium. In anotherembodiment, the sample being subjected to analysis is labeled with afluorescent molecule. In another embodiment, samples are radioactivelylabeled and detected with a radiation scanning device. In addition, theycan be detected by silverstaining, Coomassie blue staining, and bybinding of other proteins (e.g., antibodies).

[0146] In another embodiment, a stack of one or more gel- orliquid-filled microhole arrays can be formed to simulate an array ofcapillaries. An array containing a plurality of reactions can be placedupon the stack, and the reactions electrophoresed out of the array andinto the stack. Subsequent to electrophoresis, the stack can bedisassembled and the presence of a molecule in a particular array can becorrelated with the position (level) of that array in the stack. Thethickness of each array in the stack need not be uniform, although, inone embodiment, the stack comprises a plurality of arrays of uniformthickness. The holes in the stack can be filled with agarose,acrylamide, or any other medium suitable for electrophoresis. A similarstacking format can be used, for example, to conduct unit gravitysedimentation through a liquid.

[0147] Kits

[0148] Included within the embodiments of the invention are kitscomprising the apparatus for containing multiple micro-volume liquidsamples as described Supra. The kits may also comprise a component of areaction to be carried out in the apparatus; the component may be eithera reactant or a reagent. In some embodiments, the reactant and/orreagent may be contained within one or more microholes of the apparatus.The kit may also contain in addition to the microhole apparatus, one ormore hydrophobic substances to be used with the apparatus. Thehydrophobic substances may be a hydrophobic fluid and/or a solidhydrophobic cover (e.g., a teflon porous membrane and/or evaporationseals (e.g. optically clear silicone sheets). Additional contents of thekit may be a chamber for maintaining the appropriate environmentalconditions, e.g., humidity and/or temperature, for the reaction(s) thatare to be carried out using the microhole apparatus, and an apparatus(s)for loading the samples into the sample chambers. When the contents ofthe kit include a fluid substance, the fluid will be packaged in anappropriate container. Desirably, the kit will also contain instructionsfor use of the microhole apparatus.

[0149] The invention is further illustrated by the following nonlimitingexamples.

EXAMPLES Example 1

[0150] PCR-mediated Analysis of CAG Repeat Length in the Human hSK Gene

[0151] A 10 ml aliquot of 2× PCR mastermix is prepared from commerciallyavailable components (e.g. GeneAmp® PCR Reagent Kit with AmpliTaq® DNAPolymerase; PE Biosystems, Foster City, Calif.) and customoligonucleotide primers.

[0152] The master mix contains each of the following components at1.6-times the desired final concentration:

[0153] Deoxynucleotides (dATP, dCTP, dGTP, TTP (or UTP))

[0154] A forward primer: FrwCAG2: GGA CCC TCG CTG CAG CCT CA

[0155] A reverse primer: RewCAG2: GCA AGT GGT CAT TGA GAT TGA GCT GCC T

[0156] A thermostable DNA polymerase (e.g. AmpliTaq® DNA Polymerase)

[0157] A buffer

[0158] MgCl₂

[0159] Using a Hamilton 4000 robot, 1.7 μl of mastermix is dispensedinto each of 576 microholes (24 rows and 24 columns) in a solidsubstrate (the microhole apparatus). The holes occur in the shape of aright circular cylinder of 1.2 mm diameter and 1.6 mm height. Themicrohole apparatus is suspended such that its lower face is shallowlyimmersed in mineral oil at a depth such that no mineral oil is forcedinto the microholes. Immediately following dispensing of the master mix,1 μl of mineral oil is dispensed on top of each microhole.

[0160] Alternatively the apparatus is touched or immersed in a reservoirof master mix so that each microhole is filled with mastermix (internalvolume of 1.7 μl). The apparatus is then shallowly immersed in mineraloil so that it is submerged to a depth of <1 mm.

[0161] Template DNA is prepared from blood or other tissue(s) from humanpatients of interest e.g., using QIAamp 96 DNA Blood BioRobot Kit(QIAGEN Inc., Valencia, Calif.). Using a Hamilton 4000 robot, 1 μl ofDNA is removed from its position in a 96-well dish and is pipettedthrough the mineral oil into each microhole. Each microhole contains adistinct template DNA sample (derived from an individual patient) buteach patient sample can be assayed multiple times in differentmicroholes.

[0162] Following dispensing of the template DNA, the apparatus isimmersed in a small volume (1-5 ml) of mineral oil. The mineral oil isthermally cycled as follows:

[0163] 94° C. for 40 seconds, 4 cycles of (94° C. for 10 seconds, 70° C.for 50 seconds), and 28 cycles of (94° C. for 10 seconds, 68° C. for 50seconds). After thermocycling, the substrate is removed from the oilbath and loading dye (which can contain bromphenol blue, xylene cyanoleFF and/or Ficoll (commercially available from e.g., Bio101, Inc.,Carlsbad, Calif.)) is dispensed into each microhole either using aHamilton 4000 robot or by hand using an adjustable distance multichannelpipettor such as the Matrix Impact EXP. The material in the microholesis then aspirated and samples are dispensed into the wells of an 8%polyacrylamide gel and are electrophoresed to resolve differently sizedproducts.

Example 2

[0164] Expression Analysis

[0165] Template mRNA (or total RNA) is prepared from the tissue(s) ofhuman patients of interest e.g. using an RNeasy 96 BioRobot Kit (QIAGENInc., Valencia, Calif.). The RNA is combined with a mix containingreagents for reverse transcription and PCR amplification (e.g., GeneAmp®Gold RNA PCR Reagent Kit, PE Biosystems, Foster City, Calif.), excludingsequence-specific oligonucleotides and probes, such that the reagentsare present at 1.6 times the desired final concentration. 1.7 μl oftemplate/reagent mix is dispensed into each of 576 microholes in amicrohole apparatus (the apparatus, as described above). Immediatelyfollowing dispensing of the template, 1 μl of mineral oil is dispensedon top of each microhole.

[0166] Alternatively, the apparatus is touched or immersed in areservoir of template RNA/reagent mix so that each microhole is filledwith RNA/reagent mix (volume 1.7 μl). The substrate is then shallowlyimmersed in mineral oil so that it is submerged to a depth of <1 mm.

[0167] PCR primer/probe combinations corresponding to genes of interestare designed using e.g. Primer Express software (PE Biosystems, FosterCity, Calif.). The fluorogenic probe for each sequence consists of anoligonucleotide with both reporter and quencher dye attached. Each probeanneals specifically between the forward and reverse amplificationprimers. When the probe is cleaved by the 5′ nuclease activity of a DNApolymerase (e.g. AmpliTaq® DNA Polymerase; PE Biosystems, Foster City,Calif.), the reporter dye is separated from the quencher dye and asequence-specific fluorescrent signal is generated. The fluorescenceintensity of the dye is proportional to the amount of starting materialpresent in a patient sample.

[0168] Cognate PCR primers and probes corresponding to each gene ofinterest are mixed together at 2.7-fold the desired final concentration.1 μl of each of the individual primer/probe mixes are dispensed into themicroholes containing the RNA template/reagent mix using a Hamilton 4000robot. Each microhole contains a distinct primer/probe combination(corresponding to an individual gene) but each gene can be assayedmultiple times using different microholes.

[0169] Alternatively, primers and probes can be prepared at 2-fold thedesired final concentration and can be dispensed into empty microholesof a separate apparatus. The primer and probe mixes are introduced intothe RNA template/reagent mix by touching the two apparatuses together toallow mixing of the reagents.

[0170] Alternatively the desired amount of primers and probes can bedispensed into empty microholes, following which the apparatus isdessicated, allowing the primers/probe mix to dry onto the wall of eachmicrohole. The entire apparatus is then touched or immersed in areservoir of template RNA/reagent mix, so that each microhole is filledwith RNA/reagent mix (volume 1.7 μl) as described above. The RNA/reagentmix rehydrates the dessicated primer/probe mix.

[0171] Following the introduction of template DNA, the substrate isimmersed in a small volume (1-5 ml) of mineral oil. The mineral oil isthermally cycled as follows:

[0172] 95° C. for 10 minutes, 20-40 cycles of (95° C. for 15 seconds,60° C. for 60 seconds). After thermocycling, the apparatus is removedfrom the oil bath, covered on both sides with microscope slidecoverslips and scanned on a confocal microscope. The substrate can bereturned to the oil bath for additional cycling after scanning, ifdesired. Alternatively a device can be used to scan the reaction duringeach cycle of PCR while cycling is occurring.

Example 3

[0173] Microhole PCR with Pre-affixed Oligonucleotide Primers andin-situ Fluorescent Detection

[0174] PCR amplification of Lambda phage was performed in a microholearray with hole diameters of 1.0 mm and a titanium plate thickness of1.1 mm. The titanium plate 1 was wrapped at the ends with aluminum tape71 (FIG. 7) to ensure thermal contact of the plate to the heat block ofa modified Perkin-Elmer 480 (PE480) thermal cycler while preventingphysical contact between the heat block and the reactions contained inthe holes. The PE480 thermal cycler was modified by cutting a channelacross the width of the heater block to allow insertion and thermalcycling of microhole array plates. The channel was 2.4 mm wide, 18.8 mmdeep and cut across the entire face of the heat block, a distance of 90mm. The open ends of the channel were filled with heat conductivesilicone caulk (Ultra Copper, Loctite Corporation, Rocky Hill, Conn.)and the cured assembly was filled with laboratory grade mineral oil(Sigma Chemical #M-5904).

[0175] Primers were designed to amplify a 632 bp region of the LambdaDNA, forward primer=tggtatgaccggcatcct, reverseprimer=tcggcgtgtcatatttcact. Initial loading of primer pairs onto thedried titanium microhole plate was 0.5 μl of 10 μmolar dilutions, giving5 picomoles of forward and reverse primer in each well. The loadedreaction assembly was placed onto a 95° C. hot plate, the aluminum tapeat the ends of the titanium plate providing a thermal path for increasedevaporation rate while creating a standoff so that the loaded microholeswould not make physical contact to the surface of the hot plate. Afterseveral minutes, the dried plate was removed.

[0176] A reaction mixture of 1 μl picogreen (Molecular Probes, Eugene,Oreg.), 1 ng Lambda Control DNA (AB Gene, Surrey, UK), 3 μl TE(pH=7.5),and 5 μl 2× master mix was prepared. The 2× master mix consisted of 10μl 10× buffer concentration, 10 μl MgCl (for a final concentration of2.5 mM), 5 units RedTaq (Sigma Chemical #D-2812), 12 μl dNTPs (1.25micromolar of each A,C,G,T). 0.5 μl of this reaction mixture was addedto each of the holes in the microhole array, and thermal cycling wasperformed.

[0177] Thermal cycling parameters for the PE480 were as follows: 3minutes at 95° C., 30 cycles of 95° C. for 30 secs, 55° C. for 30 secs,73° C. for 60 secs, and 10 minutes at 72° C. After thermal cycling themicrohole array was removed from the thermal cycler and placed on aconfocal laser scanner/imager without removing the oil from the titaniumsubstrate. The in-house imager/scanner uses a pair of coupledscrew-drive robotic translation stages to scan the microhole arraysubstrate in the x- and y-directions. An Argon-ion laser is focusedthrough an objective lens onto the substrate, the excited emissionreturns through the objective lens and dichroic mirrors direct light of530 nm (+/−20 nm) wavelength and 570 nm (+/−20 nm) wavelength to twoseparate photomultiplier tubes (PMTs). The signal of the PMTs iscollected and displayed on the attached computer.

[0178] The argon-ion laser excited the picogreen (which stronglyfluoresces in the presence of double stranded DNA, but fluoresces onlyweakly in the presence of RNA and single stranded DNA) at 488 nm. Afluorescent signal from these reactions which is higher thanpre-calibrated background signals indicates successful rehydration ofthe primers and amplification of the Lambda template.

[0179] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity ofunderstanding, it will be apparent to those skilled in the art thatvarious changes and modifications can be practiced without departingfrom the spirit of the invention. Therefore the foregoing descriptionsand examples should not be construed as limiting the scope of theinvention.

What is claimed is:
 1. A method for simultaneously conducting aplurality of micro-volume reactions, the method comprising: (a)introducing a plurality of liquid samples into the sample chambers of amicrohole apparatus, wherein the samples contain necessary reactioncomponents; and (b) placing the apparatus into an environment favorableto the reaction; wherein the microhole apparatus comprises a substrate,wherein the substrate defines a plurality of sample chambers, whereineach sample chamber: (i) extends through the substrate; (ii) comprisesone or more walls and an opening at each end; and holds a sample suchthat the sample is in the form of a thin film such that a liquid samplepresent in one sample chamber does not intermix with a liquid samplepresent in another sample chamber; and wherein the sample chamber has aheight to width ratio of less than 1:1, wherein the height of the samplechamber is measured from one face of the substrate to the other.
 2. Themethod of claim 1 , wherein the apparatus is substantially free ofcontaminating amplifiable polynucleotides.
 3. The method of claim 1 ,wherein the reactions are selected from the group consisting of ligationreactions, primer extension reactions, nucleotide sequencing reactions,restriction endonuclease digestions, biological interactions,oligonucleotide synthesis reactions, and polynucleotide hybridizationreactions.
 4. The method of claim 3 , wherein the biologicalinteractions are selected from the group consisting of avidin-biotininteractions, streptavidin-biotin interactions, antigen-antibodyinteractions, hapten-antibody interactions and ligand-receptorinteractions.
 5. The method according to claim 1 wherein the environmentis selected from the group consisting of a hydrophobic medium and ahumidified chamber.
 6. The method according to claim 1 , wherein resultsof the reactions are monitored.
 7. The method according to claim 6wherein the results are monitored by a method selected from the groupconsisting of optical monitoring, mass spectrometry and electrophoresis.8. The method according to claim 1 , wherein progress of the reactionsare monitored during the course of the reactions.
 9. The methodaccording to claim 1 , wherein one or more of the reactions aresupplemented with one or more reagents during the course of thereaction.
 10. The method according to claim 1 , wherein a reactioncomponent is affixed to the substrate.
 11. The method according to claim10 wherein the environment is selected from the group consisting of ahydrophobic medium and a humidified chamber.
 12. A method for adding acomponent to a micro-volume reaction, wherein the method comprises thesteps of: (a) providing a first apparatus comprising a first samplechamber containing a reaction mixture; (b) providing a second apparatuscomprising a second sample chamber containing the component; and (c)bringing the apparatuses into proximity such that liquid contact isestablished between the first sample chamber and the second samplechamber; wherein each apparatus comprises a substrate, wherein thesubstrate defines a plurality of sample chambers, wherein each samplechamber: (i) extends through the substrate; (ii) comprises one or morewalls and an opening at each end; and (iii) holds a sample such that thesample is in the form of a thin film such that a liquid sample presentin one sample chamber does not intermix with a liquid sample present inanother sample chamber; and wherein the sample chamber has a height towidth ratio of less than 1:1, wherein the height of the sample chamberis measured from one face of the substrate to the other.
 13. The methodaccording to claim 12 wherein multiple components are added to areaction, by providing additional apparatuses, wherein each of thecomponents is present in a sample chamber of an apparatus.
 14. Themethod according to claim 13 , wherein the apparatuses are brought intoproximity such that liquid contact is established between correspondingsample chambers of the apparatuses.
 15. A method for simultaneouslyadding a component to a plurality of micro-volume reactions wherein, inthe method according to claim 12 , the apparatuses are brought intoproximity such that liquid contact is established between correspondingsample chambers of the apparatuses.
 16. The method of claim 12 , whereinthe component is a nucleic acid.
 17. A method for adding a nucleic acidto a micro-volume reaction, wherein the method comprises the steps of:(a) providing a first apparatus comprising a first sample chambercontaining a reaction mixture; (b) providing a second apparatuscomprising a second sample chamber containing the nucleic acid; and (c)bringing the apparatuses into proximity such that liquid contact isestablished between the first sample chamber and the second samplechamber; wherein each apparatus comprises a substrate, wherein thesubstrate defines a plurality of sample chambers, wherein each samplechamber: (i) extends through the substrate; (ii) comprises one or morewalls and an opening at each end; and (iii) holds a sample such that thesample is retained in the apparatus through surface tension and suchthat a liquid sample present in one sample chamber does not intermixwith a liquid sample present in another sample chamber.
 18. A method forintroducing a liquid sample into a sample chamber, wherein the methodcomprises the steps of: (a) contacting an apparatus with a liquidsolution; and (b) removing the apparatus from the solution; wherein theapparatus comprises a substrate, wherein the substrate defines aplurality of sample chambers, wherein each sample chamber: (i) extendsthrough the substrate; (ii) comprises one or more walls and an openingat each end; and (iii) holds a sample such that the sample is in theform of a thin film such that a liquid sample present in one samplechamber does not intermix with a liquid sample present in another samplechamber; and wherein the sample chamber has a height to width ratio ofless than 1:1, wherein the height of the sample chamber is measured fromone face of the substrate to the other.
 19. A method for introducing aliquid sample comprising a nucleic acid into a sample chamber, whereinthe method comprises the steps of: (a) contacting an apparatus with aliquid solution comprising a nucleic acid; and (b) removing theapparatus from the solution; wherein the apparatus comprises asubstrate, wherein the substrate defines a plurality of sample chambers,wherein each sample chamber: (i) extends through the substrate; (ii)comprises one or more walls and an opening at each end; and (iii) holdsa sample such that the sample is retained in the apparatus throughsurface tension and such that a liquid sample present in one samplechamber does not intermix with a liquid sample present in another samplechamber.
 20. A method for diluting a solution, wherein the methodcomprises the steps of: (a) providing a first apparatus comprising afirst sample chamber containing the solution (b) providing a secondapparatus comprising a second sample chamber containing a diluent; and(c) bringing the apparatuses into proximity such that liquid contact isestablished between the first sample chamber and the second samplechamber; wherein each of the apparatuses comprises a substrate, whereinthe substrate defines a plurality of sample chambers, wherein eachsample chamber: (i) extends through the substrate; (ii) comprises one ormore walls and an opening at each end; and (iii) holds a sample suchthat the sample is in the form of a thin film such that a liquid samplepresent in one sample chamber does not intermix with a liquid samplepresent in another sample chamber; and wherein the sample chamber has aheight to width ratio of less than 1:1, wherein the height of the samplechamber is measured from one face of the substrate to the other.
 21. Amethod for simultaneously diluting a plurality of solutions wherein, inthe method of claim 20 , the apparatuses are brought into proximity suchthat liquid contact is established between corresponding sample chambersof the apparatuses.
 22. The method according to claim 20 , wherein steps(b) through (c) are repeated one or more times using a new secondapparatus containing fresh solvent at each repetition of step (b).
 23. Amethod for diluting a solution comprising a nucleic acid, wherein themethod comprises the steps of: (a) providing a first apparatuscomprising a first sample chamber containing the solution whichcomprises a nucleic acid; (b) providing a second apparatus comprising asecond sample chamber containing a diluent; and (c) bringing theapparatuses into proximity such that liquid contact is establishedbetween the first sample chamber and the second sample chamber; whereineach of the apparatuses comprises a substrate, wherein the substratedefines a plurality of sample chambers, wherein each sample chamber: (i)extends through the substrate; (ii) comprises one or more walls and anopening at each end; and (iii) holds a sample such that the sample isretained in the apparatus through surface tension and such that a liquidsample present in one sample chamber does not intermix with a liquidsample present in another sample chamber.
 24. A method forsimultaneously diluting a plurality of solutions comprising nucleicacids wherein, in the method of claim 23 , the apparatuses are broughtinto proximity such that liquid contact is established betweencorresponding sample chambers of the apparatuses.
 25. The methodaccording to claim 23 , wherein steps (b) through (c) are repeated oneor more times using a new second apparatus containing fresh solvent ateach repetition of step (b).
 26. A method for selective retention of amolecule in a first sample chamber, wherein the method comprises thesteps of: (a) providing a first apparatus, wherein the first samplechamber contains a solution comprising the molecule and one or moreadditional solute molecules of higher diffusibility; (b) providing asecond apparatus comprising a second sample chamber containing asolvent; (c) bringing the apparatuses into proximity such that liquidcontact is established between the first sample chamber and the secondsample chamber; and (d) removing the apparatuses from proximity; whereineach of the apparatuses comprises a substrate, wherein the substratedefines a plurality of sample chambers, wherein each sample chamber: (i)extends through the substrate; (ii) comprises one or more walls and anopening at each end; and (iii) holds a sample such that the sample is inthe form of a thin film such that a liquid sample present in one samplechamber does not intermix with a liquid sample present in another samplechamber; and wherein the sample chamber has a height to width ratio ofless than 1:1, wherein the height of the sample chamber is measured fromone face of the substrate to the other.
 27. The method according toclaim 26 , wherein steps (b) through (d) are repeated one or more timesusing a new second apparatus containing fresh solvent at each repetitionof step (b).
 28. The method according to claim 26 wherein the method isused for desalting a solution.
 29. The method according to claim 28 ,wherein steps (b) through (d) are repeated one or more times using a newsecond apparatus containing fresh solvent at each repetition of step(b).
 30. A method for simultaneously desalting a plurality of solutions,wherein, in the method of claim 26 , the apparatuses are brought intoproximity such that liquid contact is established between correspondingsample chambers of the apparatuses.
 31. A method for selective retentionof a nucleic acid in a first sample chamber, wherein the methodcomprises the steps of: (a) providing a first apparatus, wherein thefirst sample chamber contains a solution comprising the nucleic acid andone or more additional solute molecules of higher diffusibility; (b)providing a second apparatus comprising a second sample chambercontaining a solvent; (c) bringing the apparatuses into proximity suchthat liquid contact is established between the first sample chamber andthe second sample chamber; and (d) removing the apparatuses fromproximity; wherein each of the apparatuses comprises a substrate,wherein the substrate defines a plurality of sample chambers, whereineach sample chamber: (i) extends through the substrate; (ii) comprisesone or more walls and an opening at each end; and (iii) holds a samplesuch that the sample is retained in the apparatus through surfacetension and such that a liquid sample present in one sample chamber doesnot intermix with a liquid sample present in another sample chamber. 32.The method according to claim 31 , wherein steps (b) through (d) arerepeated one or more times using a new second apparatus containing freshsolvent at each repetition of step (b).
 33. The method according toclaim 31 wherein the method is used for desalting a solution comprisinga nucleic acid.
 34. The method according to claim 33 , wherein steps (b)through (d) are repeated one or more times using a new second apparatuscontaining fresh solvent at each repetition of step (b).
 35. A methodfor simultaneously desalting a plurality of solutions comprising anucleic acid, wherein, in the method of claim 31 the apparatuses arebrought into proximity such that liquid contact is established betweencorresponding sample chambers of the apparatuses.
 36. A method forparallel electrophoretic analysis of a plurality of micro-volumereactions, wherein the method comprises: (a) conducting the reactions ina microhole apparatus; (b) placing the apparatus in contact with anelectrophoresis medium; and (c) conducting electrophoresis; wherein theapparatus comprises a substrate, wherein the substrate defines aplurality of sample chambers, wherein each sample chamber: (i) extendsthrough the substrate; (ii) comprises one or more walls and an openingat each end; and (iii) holds a sample such that the sample is retainedin the apparatus through surface tension and such that a liquid samplepresent in one sample chamber does not intermix with a liquid samplepresent in another sample chamber.
 37. The method according to claim 36, wherein the apparatus is placed with one face in contact with theelectrophoresis medium.
 38. The method according to claim 37 , whereinthe electrophoresis medium is contained within the sample chambers ofone or more additional apparatuses, wherein each additional apparatuscomprises a substrate, wherein the substrate comprises an upper face anda lower face, wherein the substrate defines a plurality of samplechambers, wherein each sample chamber: (a) extends through thesubstrate, (b) comprises one or more walls and an opening on each faceof the substrate, and (c) holds a sample in the form of a thin film;such that a liquid sample present in one sample chamber does notintermix with a liquid sample present in another sample chamber.
 39. Themethod according to claim 38 , wherein, in the additional apparatuses,corresponding sample chambers are aligned.
 40. A method for preparing aplurality of samples for mass spectrometric analysis, wherein thesamples are placed in an apparatus that comprises a substrate, whereinthe substrate defines a plurality of sample chambers, wherein eachsample chamber: (a) extends through the substrate; (b) comprises one ormore walls and an opening at each end; and (c) holds a sample such thatthe sample is retained in the apparatus through surface tension and suchthat a liquid sample present in one sample chamber does not intermixwith a liquid sample present in another sample chamber.
 41. The methodaccording to claim 40 , wherein the analysis is by matrix assisted laserdesorption ionization time-of-flight (MALDI-TOF) spectrometry.
 42. Themethod according to claim 41 , wherein the analysis is used to detect agenetic polymorphism.
 43. The method according to claim 42 whereindetection is by single base primer extension.
 44. A method for mixing aplurality of micro-volume samples, the method comprising: (a) providinga first microhole apparatus comprising a substrate, wherein thesubstrate defines a plurality of sample chambers, wherein each samplechamber: (i) extends through the substrate; (ii) comprises one or morewalls and an opening at each end; and (iii) holds a sample such that thesample is retained in the apparatus through surface tension and suchthat a liquid sample present in one sample chamber does not intermixwith a liquid sample present in another sample chamber; (b) providing asecond microhole apparatus comprising a substrate, wherein the substratedefines a plurality of sample chambers, wherein each sample chamber: (i)extends through the substrate; (ii) comprises one or more walls and anopening at each end; and (iii) holds a sample such that the sample isretained in the apparatus through surface tension and such that a liquidsample present in one sample chamber does not intermix with a liquidsample present in another sample chamber; and (c) bringing theapparatuses into proximity such that liquid contact is establishedbetween more than one sample chamber from the first apparatus and asample chamber in the second apparatus.
 45. An apparatus for containingmultiple micro-volume liquid samples comprising a substrate, wherein thesubstrate defines a plurality of sample chambers, wherein each samplechamber: (a) extends through the substrate, (b) comprises one or morewalls and an opening at each end, and (c) holds a sample such that thesample is in the form of a thin film such that a liquid sample presentin one sample chamber does not intermix with a liquid sample present inanother sample chamber; and wherein the sample chamber has a height towidth ratio of less than 1:1, wherein the height of the sample chamberis measured from one face of the substrate to the other.
 46. Anapparatus according to claim 45 wherein the substrate compriseshydrophobic regions, wherein the hydrophobic regions are located on thesubstrate such that a liquid sample present in one sample chamber doesnot intermix with a liquid sample present in another sample chamber. 47.An apparatus according to claim 46 , wherein the substrate comprises anupper face and a lower face.
 48. An apparatus according to claim 47 ,wherein the through axes of the sample chambers are perpendicular toboth faces of the substrate.
 49. An apparatus according to claim 48 ,wherein the sample chamber has the shape of a right circular cylinder.50. An apparatus according to claim 48 , wherein the sample chamber hasthe shape of a right polygonal prism.
 51. An apparatus according toclaim 46 , wherein the hydrophobic regions are located on the upper andlower faces of the substrate such that the openings of at least onesample chamber from at least one adjacent sample chamber by ahydrophobic region.
 52. An apparatus according to claim 51 , whereinadditional hydrophobic regions are located on the walls of the samplechambers.
 53. An apparatus according to claim 46 , wherein hydrophobicregions are located on the walls of the sample chambers.
 54. Anapparatus according to claim 53 , wherein the hydrophobic region formsan annular ring along the wall of the sample chamber.
 55. An apparatusaccording to claim 53 , comprising two or more hydrophobic regions, eachforming an annular ring along the wall of the sample chamber, whereinthe hydrophobic regions define one or more annular non-hydrophobic ringstherebetween.
 56. An apparatus according to claim 45 farther comprisingat least one component of a reaction to be carried out in the apparatus.57. An apparatus according to claim 45 wherein a reaction component isaffixed to the substrate.
 58. An apparatus according to claim 45 ,wherein the component is a reagent used in a nucleotide sequencingreaction.
 59. An apparatus according to claim 45 wherein the componentis one used in a hybridization reaction.
 60. An apparatus according toclaim 46 , wherein the component is a reagent used in a nucleotidesequencing reaction.
 61. An apparatus according to claim 46 wherein thecomponent is one used in a hybridization reaction.
 62. An apparatusaccording to claim 45 , wherein the apparatus is substantially free fromcontaminating amplifiable polynucleotides.
 63. A kit comprising anapparatus for containing multiple micro-volume liquid samples comprisinga substrate, wherein the substrate defines a plurality of samplechambers, wherein each sample chamber: (a) extends through thesubstrate, (b) comprises one or more walls and an opening at each end,and (c) holds a sample such that the sample is in the form of a thinfilm such that a liquid sample present in one sample chamber does notintermix with a liquid sample present in another sample chamber; andwherein the sample chamber has a height to width ratio of less than 1:1,wherein the height of the sample chamber is measured from one face ofthe substrate to the other; and further comprising a reaction componentpackaged in a suitable container.
 64. The kit according to claim 63 ,wherein the reaction component is a reagent for performing a reactionselected from the group consisting of ligation reactions, primerextension reactions, nucleotide sequencing reactions, restrictionendonuclease digestions, oligonucleotide synthesis, hybridizationreactions and biological interactions.
 65. A kit according to claim 63 ,further comprising a hydrophobic substance to be used with theapparatus.
 66. A kit according to claim 65 , wherein the hydrophobicsubstance is a hydrophobic fluid packaged in a suitable container.
 67. Akit according to claim 65 , wherein the hydrophobic substance is ahydrophobic cover.
 68. A kit according to claim 63 , further comprisinga chamber for maintaining the appropriate environmental conditions for areaction to be carried out in the apparatus.
 69. A kit according toclaim 63 , further comprising an apparatus for loading samples into thesample chambers.